WO1997001352A1 - Compositions et procedes utilisant la glycoproteine associee a la myeline (mag) et ses inhibiteurs - Google Patents

Compositions et procedes utilisant la glycoproteine associee a la myeline (mag) et ses inhibiteurs Download PDF

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Publication number
WO1997001352A1
WO1997001352A1 PCT/US1996/011058 US9611058W WO9701352A1 WO 1997001352 A1 WO1997001352 A1 WO 1997001352A1 US 9611058 W US9611058 W US 9611058W WO 9701352 A1 WO9701352 A1 WO 9701352A1
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mag
growth
cells
pharmaceutical composition
neurite
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PCT/US1996/011058
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English (en)
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Marie T. Filbin
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Research Foundation Of Cuny, Hunter College
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Priority to AU64012/96A priority Critical patent/AU731044B2/en
Priority to DE69633336T priority patent/DE69633336T2/de
Priority to JP50458997A priority patent/JP4841017B2/ja
Priority to EP96923524A priority patent/EP0835127B1/fr
Priority to AT96923524T priority patent/ATE275415T1/de
Publication of WO1997001352A1 publication Critical patent/WO1997001352A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7016—Disaccharides, e.g. lactose, lactulose
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to the novel
  • MAG myelin-associated glycoprotein
  • this invention relates to compositions and methods useful for reversing inhibition of neural
  • compositions according to this invention are provided. regulating and for promoting neural growth or regeneration in the nervous system, methods for treating injuries or damage to nervous tissue or neurons, and methods for treating neural degeneration associated with disorders or diseases, comprising the step of administering at least one of the compositions according to this invention are
  • the mammalian nervous system does not regenerate after injury despite the fact that there are many molecules present which encourage/promote axonal (nerve) growth. It is believed that the lack of regeneration caused by the presence of molecules in the central nervous system (CNS) and the peripheral nervous system (PNS) which actively prevent/inhibit regeneration. Hence, the well documented inability of the adult mammalian CNS to regenerate after injury is believed to result from a predominance of
  • myelin-specific inhibitory molecules can largely account for the lack of CNS regeneration and their identification will help in the design of therapies to encourage regrowth after injury. The precise molecules responsible for this inhibition have, so far, remained elusive. If these inhibitory molecules can be identified and blocked, then neural regeneration can be encouraged.
  • inhibitory action of these two protein fractions is that antibodies raised to proteins eluted from these regions of polyacrylamide gels after separation of CNS myelin
  • collapsins In addition to inhibitory molecules in myelin, another family of proteins has recently been identified whose members inhibit axonal regeneration. These molecules are called collapsins (Luo et al., Cell, 75, pp. 217-27 (1993)). However, collapsins are found ubiquitously throughout the nervous system and as they are found in regions of the nervous system in which axons will grow, i.e. gray matter, they are unlikely to contribute
  • collapsins most likely play a role in guiding growing axons during development.
  • MAG like many members of the Ig-superfamily of molecules, could promote neurite outgrowth, in this case, from dorsal root ganglion (DRG) neurons from 2 day old rats (13).
  • DRG dorsal root ganglion
  • cerebellar neurons from rats of all ages up to adult cerebellar neurons from rats of all ages up to adult.
  • MAG specifically block both stimulatory and inhibitory effects of MAG on neurite outgrowth.
  • MAG therefore, depending on the age and the type of neuron, can either promote or inhibit neurite outgrowth.
  • MAG is an inhibitor of axonal growth (McKerracher et al., Neuron, 13, pp. 805-811 (1994); WO 95/22344 (24 August 1995);
  • inhibitory the precise nature of these components has not been identified, i.e., they have not been cloned nor have the proteins been purified. In addition, there was no information available on the component on the neuron that the putative inhibitory molecules interact with to prevent regrowth. As no inhibitory nor interacting molecules had been precisely identified, it was difficult, if not
  • the present invention solves the problems referred to above by identifying MAG as a potent inhibitor of axonal regeneration in the central nervous system (CNS) and the peripheral nervous system (PNS).
  • the present invention provides compositions and methods for blocking or manipulating the levels of MAG activity in the nervous system.
  • compositions comprise a pharmaceutically acceptable carrier and a therapeutically effective amount of at least one inhibitor of MAG.
  • Inhibitors of MAG include but are not limited to anti-MAG antibodies, altered and/or mutated forms of MAG
  • sialic acid-bearing sugars characterized by an altered biological activity, free sialic acid-bearing sugars, modified derivatives of sialic acid attached to a sugar, a sialic acid-bearing sugar attached to a protein or lipid carrier molecule, a modified sialic acid-bearing sugar attached to a protein or lipid carrier molecule and a sialic acid glycopeptide.
  • the MAG inhibitor comprises a small sialic acid-bearing oligosaccharide
  • sialic acid analog is sialo 2,3- ⁇ lactose (2,3-SL) or 2,3-dideoxy sialic acid (DD-NANA).
  • the MAG inhibitor comprises an altered and/or mutant form of MAG which can inhibit the binding of endogenous MAG to neurons in the CNS or PNS.
  • Altered forms of MAG preferably
  • MAG-Fc human immunoglobulin molecule
  • Preferred altered/mutated forms of MAG are soluble molecules which harbor one or more mutations in the MAG molecule that reduce or eliminate its ability to inhibit or promote neurite outgrowth compared to endogenous MAG or MAG-Fc, but do not significantly diminish the binding of the altered or mutant form of MAG to neuronal surfaces.
  • Most preferred altered/mutant forms of MAG are soluble molecules comprising a truncated form of MAG-Fc consisting of the first three of the five extracellular Ig-like domains of MAG fused to an immunoglobulin Fc domain ("MAG(d1-3)-Fc").
  • compositions comprise a therapeutically effective amount of an enzyme that can alter or remove sialic acid residues having a
  • compositions of this embodiment comprise sialidase (a neuraminidase) and sialyl transferases that alter the structure and/or lower the effective concentration of
  • the present invention also provides methods for regulating and for promoting neural growth or regeneration in the nervous system, methods for treating injuries or damage to nervous tissue or neurons, and methods for treating neural degeneration associated with disorders or diseases, comprising the step of administering at least one of the pharmaceutical compositions according to this invention.
  • the present invention provides an assay for determining whether neurite outgrowth from a particular type of neuron at a particular age is stimulated or
  • the method comprises the steps of:
  • c) comparing the relative amount of neurite growth in the cultured cells of a) and b); wherein when the relative growth of neurites in the cultured cells of a) is greater than in b), the neuronal cell is inhibited by the presence of MAG, and when the relative growth of neurites in the cultured cells of a) is less than in b), the neuronal cell is stimulated by the presence of MAG.
  • the growth-permissive substrate in the absence of MAG comprises a monolayer of mammalian cells that do not express cell-surface MAG
  • the growth-permissive substrate comprising bound MAG comprises a monolayer equivalent mammalian cells engineered to express cell surface MAG.
  • the mammalian cells are CHO cells engineered to express cell surface MAG, such as CHO-MAG2 cells.
  • the present invention also provides methods for identifying a MAG-dependent neurite growth altering agent, i.e., an agent which alters neurite outgrowth from a selected neuronal cell type, or population of mixed cell types, in the presence of MAG compared to the absence of MAG.
  • the method comprises the steps of:
  • d) comparing the relative amount of neurite growth in the cultured cells of a) and b); wherein an agent that changes the relative growth of neurites in the cultured cells of a) and b) is identified as a MAG-dependent neurite growth altering agent.
  • the growth-permissive substrate in the absence of MAG comprises a monolayer of mammalian cells that do not express cell-surface MAG
  • the growth-permissive substrate comprising bound MAG comprises a monolayer equivalent mammalian cells engineered to express cell surface MAG.
  • the mammalian cells are CHO cells engineered to express cell surface MAG, such as CHO-MAG2 cells.
  • the method for identifying a MAG-dependent neurite growth altering agent comprises the steps of:
  • the growth-permissive substrate lacking MAG comprises a monolayer of mammalian cells that do not express cell-surface MAG, such as COS or NIH 3T3 cells.
  • the growth-permissive substrate lacking MAG comprises an immobilized monolayer of a purified, growth-promoting factor.
  • One most preferred neuronal growth-promoting factor which may be immobilized onto a monolayer is the L1 glycoprotein.
  • the soluble form of MAG is a MAG-Fc fusion protein
  • the soluble control protein lacking MAG activity is a MUC 18-Fc fusion protein
  • Preferred traceable fusion proteins are
  • Fig. 1 Inhibition of Neurite outgrowth from cerebellar Neurons by MAG. Cerebellar neurons from post-natal day (PND) 1, 4 and 7 were grown overnight on a monolayer of MAG-expressing (clear bars) or control transfected CHO cells (hatched bars). Neurons were stained for GAP-43 antigen and neurite length was measured and the average length calculated from at least 150 measurements (+/- SEM).
  • Fig. 4 sialic acid-dependent Binding of MAG to Cerebellar and DRG Neurons. Radiolabelled MAG-Fc (solid bars) was allowed to bind to PND 1 cerebellar and DRG neurons.
  • Results represent the average neurite length ( ⁇ m) from at least 150 neurons +/- SEM.
  • Fig. 7 (a). Soluble MAG-Fc Inhibits Axonal Regeneration of Cerebellar Neurons Grown on L1 in a Concentration-dependent Manner. L1-Fc was immobilized and isolated PND2 cerebellar neurons were grown overnight in the presence of various concentrations of MAG-Fc (diamonds) or MUC-Fc
  • Results represent the average neurite length ( ⁇ m) +/- SEM.
  • MAG-Fc Inhibits Axonal Growth in a Specific, Sialic Acid-dependent Manner. Cerebellar neurons were grown on immobilized L1 as a substrate. MAG-Fc (column 1) or MUC-Fc (column 2) were added at a concentration of 50 ⁇ g/ml. Anti-MAG 513 monoclonal antibodies were included at a concentration of 5 ⁇ g/ml (column 3) or desialyated neurons were used (column 4). Neurite length ( ⁇ m) was measured as described in Fig. 1. Results represent the average neurite length ( ⁇ m) +/- SEM. Fig. 7(c).
  • MAG-Fc Inhibits Axonal Growth from Neurons Grown on Fibroblasts.
  • Isolated PND 2 cerebellar neurons were grown on a substrate of fibroblasts (3T3 cells) in the presence of 50 ⁇ g/ml MAG-Fc (Column 1, MAG), 50 ⁇ g/ml of MUC18-Fc (column 2, MUC 18) or in the presence of 5 ⁇ g/ml anti-MAG 513 monoclonal antibodies (column 3, anti-MAG).
  • Neurite length ( ⁇ m) was measured as described in Fig. 1. Results represent the average neurite length ( ⁇ m) +/- SEM.
  • MAG(d1-3)-Fc Binds to Neurons in a specific, Sialic Acid-dependent Manner. Cerebellar (PND2) neurons vitally labeled with fluorescein were allowed to bind to immobilized MAG(d1-5)-Fc (dark bars), MAG(d1-3)-Fc (hatched bars) or MUC18-Fc (speckled bars), in the presence
  • Cerebellar neurons were grown on MAG-expressing (MAG cells) or control ⁇ CHO cells, in the presence (+MAG1-3) or absence of MAG(d1-3)-Fc. Neurite length ( ⁇ m) was measured as described in Fig. 1. Results represent the average neurite length ( ⁇ m) +/- SEM.
  • MAG derivative refers to a molecule comprising at least one MAG extracellular domain, wherein the MAG molecule has been altered (e.g., by recombinant DNA techniques to make chimera with portions of other molecules fused to the MAG molecule, or by chemical or enzymatic modification) or mutated (e.g., internal deletions, insertions, rearrangements and point mutations). MAG derivatives, unless otherwise noted, retain MAG activity.
  • MAG bioactivity and "MAG biological activity” refer to the ability of a molecule, especially an altered or mutant form of MAG, to inhibit or promote neurite outgrowth of a selected neuronal cell type of a particular age, as detected in a neurite outgrowth assay such as those described herein, in qualitatively the same direction as cell-surface or soluble MAG.
  • MAG binding activity refers to the ability of a molecule, especially an altered or mutant form of MAG, to compete with cell-surface MAG or soluble MAG for sialic-acid dependent neuron binding in an assay such as those described herein.
  • preferred inhibitors of MAG retain MAG binding activity but have reduced or absent MAG bioactivity.
  • MAG activity refers generically to MAG bioactivity and binding activity as described above.
  • modified derivative of sialic acid refers to a sialic acid residue that has been modified chemically or enzymatically, especially to add or exchange chemical groups or side chains onto reactive positions of the molecule.
  • Sialic acids are a family of nine-carbon acidic sugars which are derivatives of neuraminic acid and which are often at the termini of cell-surface
  • NeuroAc stands for N-acetylneuraminic acid
  • GalNAc stands for N-acetylgalactosamine
  • MAG had been shown previously to promote neurite extension from particular types of neurons.
  • MAG was believed to be involved in the
  • the present invention demonstrates a novel role for MAG as an inhibitor of axonal outgrowth and hence of nerve regeneration in the central nervous system (CNS) and the peripheral nervous system (PNS).
  • CNS central nervous system
  • PNS peripheral nervous system
  • Selected neuronal cell types may be isolated from animals at increasing times in postnatal days (PND) according to the procedures described in Example 1.
  • Neurons representing a single cell type may be isolated and tested alone, or if desired, mixed populations of cells comprising one or more neuronal cell types in the presence or absence of non-neuronal cells may also be tested.
  • the present invention provides an in vitro assay for determining whether neurite outgrowth from a particular type of neuron at a particular age is stimulated or
  • Isolated neurons of choice may be cultured on a monolayer comprising a growth-permissive substrate in the presence or absence of bound MAG, and comparative neurite outgrowth may be measured.
  • the growth-permissive substrate comprises mammalian fibroblast cells which have been engineered to express MAG on their cell surfaces.
  • MAG-expressing cells may be engineered using the procedures described in Example 2. Neurite outgrowth on MAG-expressing cells may then be compared to neurite outgrowth on control cells that do not express cell-surface MAG.
  • MAG is a potent inhibitor of axonal growth from cerebellar neurons from all ages of rats tested, newborn to adult. This was determined by
  • DRG dorsal root ganglia
  • MAG inhibits axonal outgrowth from many types of neurons
  • RG Isolated retinal ganglion
  • HN hippocampal
  • MN motor neurons
  • SCG superior-cervical ganglion
  • MAG likely plays an important role in the lack of neural regeneration in all areas of the nervous system tested to date.
  • Example 2 is an effective assay whereby both the inhibition and promotion of neurite outgrowth by MAG can be monitored and characterized.
  • This assay can also be used to screen and identify agents that can block (or enhance) MAG bioactivity, thereby altering its inhibition or stimulation of axonal outgrowth in the nervous system (Example 3). Such agents are called herein "MAG-dependent neurite growth altering agents.”
  • MAG inhibits axonal outgrowth by binding to a sialic acid-bearing glycoprotein on neurons
  • MAG binds to all types of neurons tested in a sialic acid-dependent fashion (Kelm et al., Curr. Biol., 4, pp. 965-72 (1994)).
  • Fig. 4 shows the results of an aqueous MAG-Fc neuron binding assay which was performed essentially as described in Kelm et al. This experiment confirms that the binding of MAG to isolated PDN1 cerebellar neurons (whose outgrowth is inhibited by MAG) is abolished either by inclusion of anti-MAG monoclonal antibody 513 or by
  • Sialidase is an enzyme which removes sialic acid from glycoconjugates. Similarly, the binding of MAG to isolated PDN1 DRG neurons (whose outgrowth is promoted by MAG) is inhibited by inclusion of anti-MAG monoclonal antibody 513 and to as lesser extent, by sialidase
  • Axonal outgrowth assays such as those described in Example 2 were also performed in the presence of small, free sialic acid-bearing sugars. These sugars can compete with the sialic acid components of the neuronal surface for MAG binding and thereby block the inhibition (TABLE 1) or promotion (TABLE 2) of neurite growth by MAG.
  • Inclusion of increasing concentrations of either of the small sialic acid-bearing sugars 2,3-dideoxy sialic acid (DD-NANA) or sialo 2,3- ⁇ lactose (SL) reversed the inhibition of axonal growth by MAG by between 40-56% (TABLE 1) and abolished the promotion of neurite outgrowth by MAG completely (TABLE 2).
  • Neurite outgrowth was compared for cerebellar neurons from PND 2 animals, grown on MAG-expressing and control CHO cells as describe in Example 3.
  • Inhibitors of MAG binding activity include but are not limited to anti-MAG antibodies, free sialic acid-bearing sugars, modified derivatives of sialic acid
  • sialic acid-bearing sugar attached to a protein or lipid carrier molecule
  • a modified sialic acid-bearing sugar attached to a protein or lipid carrier molecule
  • sialic acid glycopeptides or glycoproteins As shown above, inhibitors of MAG binding
  • activity also include enzymes that can alter or remove sialic acid residues, especially those having a
  • Neu5Aca2 ⁇ 3GalB1 ⁇ 3GalNAc (3-O) structure which mediates MAG binding to neuronal surfaces in the PNS or CNS.
  • Preferred compositions of this embodiment comprise sialidase (a neuraminidase) and sialyl transferases that alter the structure and/or lower the effective concentration of Neu5Aca2 ⁇ 3GalB1 ⁇ 3GalNAc ("3-O") sialyated glycans. Identifying MAG-dependent Growth Regulating Agents
  • a test agent is identified as a MAG inhibitor when it promotes neurite growth from a cell type inhibited by MAG or inhibits neurite growth from a cell type stimulated by MAG.
  • an agent is a MAG agonist when it promotes neurite growth from a cell type stimulated by MAG or inhibits neurite growth from a cell type inhibited by MAG.
  • soluble MAG is a potent inhibitor of axonal regeneration
  • This invention provides a second method for assaying the effects of MAG on axonal growth and for identifying MAG-dependent neurite growth altering agents.
  • the method involves culturing separate samples of a selected neuronal cell type on a growth-permissive
  • the growth-permissive substrate lacking MAG comprises a monolayer of mammalian cells that do not express cell-surface MAG, such as COS or N1H 3T3 cells.
  • cell-surface MAG such as COS or N1H 3T3 cells.
  • the present invention is not limited by the cell types which may be employed to make such growth-permissive monolayers that do not comprise bound MAG.
  • the growth-permissive substrate lacking MAG comprises an immobilized monolayer of a purified, growth-promoting factor. It is well known in the art that neuronal cells may be cultured on growth-promoting monolayers comprising collagen or fibronectin. A preferred neuronal growth-promoting factor according to the present invention which may be immobilized onto a monolayer is the L1 glycoprotein.
  • the soluble form of MAG is a MAG-Fc fusion protein
  • the soluble control protein lacking MAG activity is a MUC18-Fc fusion protein (Example 4).
  • Preferred traceable fusion proteins are radioactively or fluorescently labeled using commercially available reagents and methods well known in the art.
  • Fig. 7a shows the results of an assay performed according to the procedures described in Example 4.
  • neurons were grown on a substrate comprising the purified growth-promoting molecule termed "L1".
  • MAG in a soluble form consisting of the extracellular domain of MAG fused to the Fc region of IgG (MAG-Fc) was added to the growing neurons (MAG-Fc; Example 4).
  • MAG-Fc As the concentration of MAG-Fc was increased, inhibition of neurite outgrowth increased, while a control chimera, MUC18-Fc, at the same concentration had no effect (Fig. 7a).
  • MAG-Fc inhibition of axonal regeneration by MAG-Fc could be reversed by either adding a monoclonal antibody directed against MAG or by desialyating the isolated neurons prior to the assay (Fig. 7b). Soluble MAG-Fc can also inhibit axonal regeneration from cerebellar neurons grown on a monolayer of fibroblasts (Fig. 7c).
  • MUC18 did not bind to neurons either in the presence or absence of anti-MAG antibodies.
  • MAG(d1-3)-Fc can compete with full-length MAG for binding to neurons because MAG(d1-3)-Fc at a concentration of 50 ⁇ g/ml can reverse by about 40% inhibition of axonal regeneration by full-length MAG expressed by CHO cells (Fig. 8c).
  • MAG-Fc a truncated form of MAG-Fc consisting of the first three of the five extracellular Ig-like domains of MAG fused to an immunoglobulin Fc domain ("MAG(d1-3)-Fc").
  • mutations may be made to MAG Ig-like domains that will also reduce or eliminate its ability to inhibit or promote neurite outgrowth without significantly diminishing the binding of the mutant form of MAG to neuronal surfaces.
  • a mutational analysis will likely lead to the identification of a localized "MAG neurite growth signaling site"
  • the MAG-dependent neurite growth regulating agents of this invention may be formulated into
  • compositions according to this invention will be useful for regulating and for promoting neural growth or regeneration in the nervous system, for treating injuries or damage to nervous tissue or neurons, and for treating neural degeneration associated with traumas to the nervous system, disorders or diseases.
  • traumas, diseases or disorders include, but are not limited to:
  • Determination of a preferred pharmaceutical formulation and a therapeutically efficient dose regiment for a given application is within the skill of the art taking into consideration, for example, the condition and weight of the patient, the extent of desired treatment and the tolerance of the patient for the treatment.
  • MAG derivatives and inhibitors of this invention may be accomplished using any of the conventionally accepted modes of administration of agents which are used to treat neuronal injuries or disorders.
  • Soluble altered and mutated forms of MAG such as those described herein are prepared from the culture media of transfected cells, e.g., COS cells (fibroblasts), transfected with expression plasmids encoding the cDNAs for these forms of MAG (Example 4).
  • the soluble MAG molecules, such as MAG-Fc, are secreted by these cells.
  • COS cells or other transfectants secreting the soluble MAG-Fc chimera may be implanted into damaged spinal cord.
  • the cells will secrete MAG-inhibiting forms of altered or mutated MAG-Fc, which prevents the endogenous MAG from interacting with the neuronal surface and thus prevents inhibition of axonal growth and
  • MAG-Fc About 2 ⁇ 10 6 transfected COS cells will secrete about 1 mg of MAG-Fc over a 5-day period. A concentration of 50 ⁇ g/ml of mutated MAG-Fc effectively reverses the inhibitory effects of wildtype MAG. Finally, within the perineurium of an adult rat spinal cord is a volume of about 0.5 ml. Therefore, if 2 ⁇ 10 6 mutated or altered MAG-Fc-secreting COS cells are implanted into an injured spinal cord, then the concentration of MAG-Fc should be maintained at about 400 ⁇ g/ml, i.e., 8-fold more
  • Transfected cells, secreting other "reversing" mutated forms of MAG or MAG "blocking" peptides can be administered to the site of neuronal injury or degeneration in a similar manner.
  • MAG inhibitors and regulators of this invention e.g., sialidases and sialyltransferases, free, protein- or lipid-attached sialic acid-bearing sugars, glycopeptides or glycoproteins, can also be used.
  • sialidases and sialyltransferases free, protein- or lipid-attached sialic acid-bearing sugars, glycopeptides or glycoproteins
  • transfected cells that secrete MAG regulating agents may be encapsulated into immunoisolatory capsules or chambers and implanted into the brain or spinal cord region using available methods that are known to those of skill in the art. See, e.g., WO 89/04655; WO 92/19195; WO93/00127; EP 127,989; U.S. Patent Nos. US 4,298,002; US 4,670,014; US 5,487,739 and references cited therein, all of which are incorporated herein by reference.
  • a pump and catheter-like device may be implanted at the site of injury to administer the agent on a timely basis and at the desired
  • compositions of this invention may be in a variety of forms, which may be selected
  • Modes of administration include, for example, solid, semi-solid and liquid dosage forms such as tablets, pills, powders, liquid solutions or suspensions, suppositories, and injectable and infusible solutions.
  • the preferred form depends on the intended mode of administration and therapeutic application.
  • Modes of administration may include oral, parenteral, subcutaneous, intravenous, intralesional or topical administration.
  • the MAG derivatives and inhibitors of this invention may, for example, be placed into sterile,
  • the MAG derivatives and inhibitors may be diluted with a formulation buffer comprising 5.0 mg/ml citric acid monohydrate, 2.7 mg/ml trisodium citrate, 41 mg/ml
  • compositions also will preferably include conventional pharmaceutically acceptable carriers well known in the art (see for example Remington's
  • Such pharmaceutically acceptable carriers may include other medicinal agents, carriers, genetic carriers, adjuvants, excipients, etc., such as human serum albumin or plasma preparations.
  • the compositions are preferably in the form of a unit dose and will usually be administered one or more times a day.
  • compositions of this invention may also be administered using microspheres, liposomes, other microparticulate delivery systems or sustained release formulations placed in, near, or otherwise in communication with affected tissues or the bloodstream.
  • sustained release carriers include semipermeable polymer matrices in the form of shaped articles such as suppositories or microcapsules.
  • Implantable or microcapsular sustained release matrices include polylactides (U.S. Patent No. 3,773,319; EP
  • Liposomes containing MAG derivatives and inhibitors can be prepared by well-known methods (See, e.g. DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci.
  • the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol.% cholesterol. The proportion of cholesterol is selected to control the optimal rate of MAG derivative and inhibitor release.
  • the MAG derivatives and inhibitors of this invention may also be attached to liposomes, which may optionally contain other agents to aid in targeting or administration of the compositions to the desired treatment site. Attachment of MAG derivatives and inhibitors to liposomes may be accomplished by any known cross-linking agent such as heterobifunctional cross-linking agents that have been widely used to couple toxins or chemotherapeutic agents to antibodies for targeted delivery. Conjugation to liposomes can also be accomplished using the carbohydrate- directed cross-linking reagent 4-(4-maleimidophenyl) butyric acid hydrazide (MPBH) (Duzgunes et al., J. Cell. Biochem. Abst. Suppl. 16E 77 (1992)). Utility of MAG Derivatives and inhibitors
  • MAG is a potent inhibitor of axonal regeneration has potential clinical use in the situations of nervous system injury ⁇ both of the
  • peripheral and central nervous systems ⁇ and in particular for CNS injury.
  • the mammalian central nervous system does not regenerate after injury even though there are many molecules present that promote and encourage a nerve to grow. The result is paralysis or brain damage. It has been shown that there are molecules present in the adult CNS that will actively prevent a nerve from regenerating. It is anticipated that if these inhibitory molecules can be first identified and subsequently blocked, then an
  • the first step is to identify what the inhibitory molecules are.
  • MAG is the first such molecule to be identified in myelin.
  • strategies can now be designed with MAG as a target such that its inhibitory function is blocked.
  • Such an agent can then be administered to damaged nerves reversing the inhibitory effects of MAG in vivo and allowing nerve regeneration to proceed.
  • the assays of the present invention are useful for identifying agents likely to reverse inhibition of nerve regeneration by MAG.
  • the inhibitory effects of MAG were shown to be blocked/prevented from functioning by agents such as sialidases or small sialic acid-bearing sugars and by soluble, mutated forms of MAG.
  • agents such as sialidases or small sialic acid-bearing sugars and by soluble, mutated forms of MAG.
  • MAG as a negative guidance cue
  • properties of MAG as a negative guidance cue can be used to guide regenerating axons to their correct target and keep them on the correct path.
  • MAG, or different domains of MAG can be administered to the precise regions of the regenerating nervous tissue to contain growth along exact pathways.
  • MAG binds to a sialic acid-bearing glycoprotein on neurons to bring about
  • sialic acid-bearing sugars and derivatives thereof can bind to MAG, prevent it from interacting with the sialic acid glycoprotein on neurons and prevent its inhibition of axonal regeneration. It is anticipated that in vivo, after injury, application of MAG inhibitors such as sialidases, free small sialic-bearing sugars or modifications of sialic acid attached to other sugars, small sialic acid-bearing sugars covalently
  • MAG mutated, soluble form of MAG
  • MAG MAG derivatives
  • MAG MAG derivatives
  • inhibitors may be used as a guidance cue in precise regions of the regenerating nervous system to keep growing axons on the correct path and moving towards the correct target.
  • Neurons were isolated essentially as described in Doherty et al., Nature, 343, pp. 464-66 (1990); Neuron, 5, pp. 209-19 (1990); and Kleitman et al., Culturing Nerve Cells, pp. 337-78, MIT Press, Cambridge, MA/London, England (G. Banker and K. Goslin, Eds.) (1991). Briefly, for animals up to nine days of age, the cerebellum, retina, hippocampus, and spinal cord were removed from two animals.
  • ganglia were removed from two animals and incubated in 5 ml of L15 medium containing 0.025% trypsin and 0.3% collagenase type I (Worthington) for 30 min. at 37°C. The ganglia were triturated with a fire-polished Pasteur pipette.
  • CHO cells deficient in the dihydrofolate reductase (dhfr) gene (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77, pp. 4216-20 (1980)) were transfected with a MAG-cDNA expression plasmid with the dhfr gene and the L-MAG cDNA in either a 5' -3' or, as a control, a 3' -5' orientation, cells with multiple copies of dhfr were selected by growing in increasing concentrations of methotrexate, and the expression of MAG by individual transfected CHO cell lines characterized as described in Mukhopadhyay et al., Neuron, 13, pp.
  • dhfr dihydrofolate reductase
  • Transfected cells were maintained in DMEM supplemented with 10% dialyzed FCS, proline (40 mg/liter), thymidine (0.73 mg/liter), and glycine (7.5 mg/liter) at 37°C in 5% CO 2 .
  • MAG2 The MAG-expressing transfected CHO cell line (“CHO-MAG2") described as MAG2 in that publication was deposited on June 27, 1996 with the American Type Culture Collection (ATCC) (Rockville, MD) according to the
  • Confluent monolayers of control and MAG-expressing CHO cells were established over a 24-hour (h) period in individual chambers of an 8-well tissue culture slide (Lab-Tek). Co-cultures were established as described previously (Doherty et al., Nature, 343, pp. 464-66 (1990); Neuron, 5, pp. 209-19 (1990); Mukhopadhyay et al., Neuron. 13, pp. 757-67 (1994)) by adding approximately 5000 cerebellar, dorsal root ganglion (DRG) and
  • SCG superior-cervical ganglion
  • Culture medium was SATO containing 2% FCS. Where indicated, 20 mU of VCS was included throughout the coculture period (see Example 4), or monolayers were incubated with small oligosaccharides for one hour before adding the neuronal cell suspension and included throughout the coculture period. After periods of time as indicated, the cocultures were fixed for 30 min with 4%
  • neuron-specific antibodies such as anti-neurofilament monoclonal antibodies, which are commercially available (e.g., Boehringer Mannheim, Sigma Immunochemicals), may be used starting at dilutions recommended by the manufacturer.
  • the appropriate species-specific, biotinylated anti-Ig secondary antibody is then selected according to the species in which the primary anti-neural antibody was generated.
  • various vital dyes e.g., Molecular Probes, Oregon
  • stain neurites may be used in this assay in place of a
  • the transfected CHO cell assay described in Example 2 may also be used to screen and identify agents that alter neurite growth properties of a particular neuronal cell type and age in a MAG-dependent fashion. Neurite outgrowth was compared for cerebellar (TABLE 1) and DRG (TABLE 2) neurons from PND 2 animals, grown on MAG-expressing and control CHO cells as described in Example 2.
  • small sialic acid-bearing sugars were included in the co-cultures at increasing concentrations. 100% inhibition was taken as the difference in length of
  • This assay may be used to test other putative MAG-dependent neurite growth regulating agents by including them in the coculture and measuring their effect in the presence and absence of cell-surface MAG as described above for the small sialic acid-bearing sugars.
  • MUC 18-Fc were transfected into COS cells and the Fc-chimeric proteins purified from the media as described in Kelm et al., Current Biol., 4, pp. 965-72 (1994) and P.R. Crocker and S. Kelm, "Methods for studying the cellular binding properties of lectin-like receptors," in Handbook of Experimental Immunology, pp. 1-30 (1995).
  • Neuron binding assays were performed essentially as described in DeBellard et al., Mol. Cell. Neuroscience, 7, pp. 89-101 (1996), which is incorporated herein by
  • Fc-chimeric proteins were adsorbed for 3 h at 37°C to wells of microtiter plates that had been coated for 2 h at 37°C with anti-human IgG at 15 ⁇ g/ml in 0.1M
  • neurons Prior to the binding assay, neurons were vitally labeled with the fluorescent dye calcein AM (Molecular Probes) by incubating 2 ⁇ 10 6 neurons in 5 ml of 10 ⁇ M calcein AM in PBS for 15 min at 37°C before being washed and resuspended in PBS.
  • fluorescent dye calcein AM Molecular Probes
  • the L1 glycoprotein is a cell adhesion molecule
  • Soluble L1-Fc chimera may be constructed using procedures known to those of skill in the art (such as those cited in Example 3; Doherty et al., Neuron, pp. 57-66 (1995), incorporated herein by reference). Soluble L1-Fc chimera, when presented to neurons, are as effective at promoting neurite outgrowth as the normal cell surface- associated L1 (Doherty et al., supra, and references cited therein which are incorporated herein by reference). As described in Doherty et al., L1-Fc chimera can stably associate with the surface of fibroblast 3T3 cells or polylysine/ collagen or polylysine/fibronectin-coated substrates.
  • Cerebellar neurons (post-natal days 2-7) were dissociated by trypsinization as described in Example 1, except that the dissociated neurons were resuspended in 5 ml of SATO medium containing 2% dialyzed FBS.
  • SATO medium containing 2% dialyzed FBS.
  • 5.0 ⁇ 10 4 cerebellar neurons were added, followed by either a single concentration (about 50 ⁇ g/ml) or increasing concentrations (e.g., 0-30 ⁇ g/ml) of MAG-Fc or MUC18-Fc chimeric soluble proteins, depending on the experiment.
  • Neurons were cultured overnight (about 16 h) at 37°C, and then fixed and stained essentially as
  • the neurite outgrowth binding assay using soluble MAG-Fc described in Example 5 may also be used to perform competitive neuron binding/growth experiments to screen and identify new agents that alter the neurite growth
  • test agent properties of a particular neuronal cell type and age in a MAG-dependent fashion.
  • concentrations of the test agent were included in the cocultures of Example 5, and the effect of the test agent in the presence and absence of soluble MAG assessed.
  • Single cell suspensions of different neurons at various postnatal ages were washed and resuspended in phosphate-buffered saline (PBS). Approximately 2 ⁇ 10 6 cells were incubated with 50 mU of Vibrio cholera sialidase (VCS, Calbiochem) (a neuraminidase) in a final volume of 0.5 ml for 2 hours at 37°C. Neurons were washed with PBS and resuspended in SATO medium containing 2% FCS for neurite outgrowth experiments, or in PBS for neurite binding assays.
  • VCS Vibrio cholera sialidase
  • This procedure may be modified by using enzymes other than sialidase that digest or otherwise modify carbohydrate structures (see, e.g., Kelm et al., Carbohydr. Res., 149, pp. 59-64 (1986), which is incorporated by reference herein).
  • sialyl transferases may be employed to alter or remove sialyated glycans on neuronal surfaces comprising sialic acid residues having a
  • COS cells transfected with an expression plasmid that encodes MAG(d1-3)-Fc were cultured and the cultures assayed for the rate of MAG(d1-3)-Fc secretion.
  • the cells secrete MAG(d1-3)-Fc, which is capable of inhibiting endogenous MAG activity in the myelin of the implant site, and neural regeneration is stimulated.

Abstract

L'invention se rapporte à une nouvelle identification de la glycoprotéine associée à la myéline ('MAG') en tant qu'inhibiteur puissant de la régénération neurale. Plus particulièrement, cette invention a trait aux compositions et aux procédés utiles pour inverser l'inhibition de la régénération neurale dans le système nerveux central et périphérique. Les dosages pour surveiller les effets de MAG sur la régénération neurale et pour identifier les agents bloquant ou favorisant les effets inhibiteurs de MAG sur l'excroissance neurale sont décrits. Des procédés de criblage pour identifier de tels agents sont également décrits. L'invention se rapporte aussi à des compositions et à des procédés utilisant des agents pouvant inverser les effets inhibiteurs de MAG sur la régénération neurale. Les procédés pour réguler et favoriser la croissance ou régération neurale dans le système nerveux, les procédés pour traiter des blessures ou des dégâts aux tissus nerveux ou aux neurones, ainsi que des procédés pour traiter la dégénération neurale associée aux troubles ou aux maladies, y compris l'administration d'au moins une des compositions selon l'invention, sont décrits.
PCT/US1996/011058 1995-06-27 1996-06-27 Compositions et procedes utilisant la glycoproteine associee a la myeline (mag) et ses inhibiteurs WO1997001352A1 (fr)

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AU64012/96A AU731044B2 (en) 1995-06-27 1996-06-27 Compositions and methods using myelin-associated glycoprotein (MAG) and inhibitors thereof
DE69633336T DE69633336T2 (de) 1995-06-27 1996-06-27 Zusamenzetzung enthaltend einen inhibitor des myelin assoziierten glycoproteins (mag), welcher einer veränderten oder mutierten form von mag enthält
JP50458997A JP4841017B2 (ja) 1995-06-27 1996-06-27 ミエリン会合糖タンパク質（ｍａｇ）およびそのインヒビターを用いた組成物および方法
EP96923524A EP0835127B1 (fr) 1995-06-27 1996-06-27 Composition comprenant un inhibiteur de la glycoproteine associee a la myeline (mag) qui contient une forme modifiee ou mutee de mag
AT96923524T ATE275415T1 (de) 1995-06-27 1996-06-27 Zusamenzetzung enthaltend einen inhibitor des myelin assoziierten glycoproteins (mag), welcher einer veränderten oder mutierten form von mag enthält

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WO2001085981A2 (fr) * 2000-05-05 2001-11-15 Research Foundation Of City University Of New York Methodes de stimulation de la regeneration et de la reparation du systeme nerveux par regulation de l'activite de l'arginase 1 et de la synthese des polyamines
AU768763B2 (en) * 1998-05-19 2004-01-08 Yeda Research And Development Co. Ltd. Activated T cells, nervous system-specific antigens and their uses
WO2004014953A2 (fr) 2002-08-06 2004-02-19 Glaxo Group Limited Anticorps
WO2006127966A2 (fr) * 2005-05-25 2006-11-30 The Johns Hopkins University Compositions et methodes servant a favoriser la regeneration de l'axone
WO2007068750A2 (fr) 2005-12-16 2007-06-21 Glaxo Group Limited Immunoglobulines
US7842666B2 (en) 2002-12-20 2010-11-30 Research Foundation Of City University Of New York Inhibitors of myelin-associated glycoprotein (MAG) activity for regulating neural growth and regeneration
US8017115B2 (en) 2003-03-19 2011-09-13 Glaxo Group Limited Therapeutical use of anti-myelin associated glycoprotein (MAG) antibodies
US8367615B2 (en) 2006-03-30 2013-02-05 Research Foundation Of City University Of New York Stimulation of neuron regeneration by secretory leukocyte protease inhibitor
US8974782B2 (en) 2011-02-07 2015-03-10 Glaxo Group Limited Treatment of stroke comprising anti-MAG antibodies

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WO1999060021A2 (fr) * 1998-05-19 1999-11-25 Yeda Research And Development Co. Ltd. Lymphocytes t actives, antigenes specifiques du systeme nerveux et leur utilisation
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WO2001085981A3 (fr) * 2000-05-05 2002-03-14 Res Foundation Of City Univers Methodes de stimulation de la regeneration et de la reparation du systeme nerveux par regulation de l'activite de l'arginase 1 et de la synthese des polyamines
US8673594B2 (en) 2000-05-05 2014-03-18 Research Foundation Of City University Of New York Methods for stimulating nervous system regeneration and repair by regulating arginase I and polyamine synthesis
WO2001085981A2 (fr) * 2000-05-05 2001-11-15 Research Foundation Of City University Of New York Methodes de stimulation de la regeneration et de la reparation du systeme nerveux par regulation de l'activite de l'arginase 1 et de la synthese des polyamines
US7741310B2 (en) * 2000-05-05 2010-06-22 Research Foundation Of The City University Of New York Methods for stimulating nervous system regeneration and repair by regulating arginase I and polyamine synthesis
WO2004014953A2 (fr) 2002-08-06 2004-02-19 Glaxo Group Limited Anticorps
US8071731B2 (en) 2002-08-06 2011-12-06 Glaxo Group Limited Humanised anti-MAG antibody or functional fragment thereof
US7612183B2 (en) 2002-08-06 2009-11-03 Glaxo Group Limited Humanised anti-mag antibody or functional fragment thereof
US7842666B2 (en) 2002-12-20 2010-11-30 Research Foundation Of City University Of New York Inhibitors of myelin-associated glycoprotein (MAG) activity for regulating neural growth and regeneration
US8017115B2 (en) 2003-03-19 2011-09-13 Glaxo Group Limited Therapeutical use of anti-myelin associated glycoprotein (MAG) antibodies
WO2006127966A3 (fr) * 2005-05-25 2007-03-01 Univ Johns Hopkins Compositions et methodes servant a favoriser la regeneration de l'axone
WO2006127966A2 (fr) * 2005-05-25 2006-11-30 The Johns Hopkins University Compositions et methodes servant a favoriser la regeneration de l'axone
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WO2007068750A2 (fr) 2005-12-16 2007-06-21 Glaxo Group Limited Immunoglobulines
US8367615B2 (en) 2006-03-30 2013-02-05 Research Foundation Of City University Of New York Stimulation of neuron regeneration by secretory leukocyte protease inhibitor
US8974782B2 (en) 2011-02-07 2015-03-10 Glaxo Group Limited Treatment of stroke comprising anti-MAG antibodies

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